Interference-suppressed immunoassay to detect anti-drug antibodies in serum samples

ABSTRACT

Herein is reported an enzyme linked immunosorbent assay for the detection of anti-drug antibodies against a drug antibody in a sample comprising a capture drug antibody and a tracer drug antibody, wherein the capture drug antibody and the tracer drug antibody are employed in a concentration of 0.5 μg/ml or more, the sample is incubated simultaneously with the capture drug antibody and the tracer drug antibody for 1 to 24 hours, the capture drug antibody and the tracer drug antibody are derivatized via a single lysine residue, the sample comprises 10% serum, and oligomeric human IgG is added to the sample prior to the incubation with the capture drug antibody and the tracer drug antibody.

Herein is reported an interference-suppressed immunoassay to detectanti-drug antibodies in serum samples from patients treated with ananti-inflammatory antibody and uses thereof.

BACKGROUND OF THE INVENTION

The clinical development of novel therapeutic antibodies requires theevaluation of their potential immunogenicity by appropriate assays(Kaliyaperumal, A. and Jing, S., Curr. Pharm. Biotechnol. 10 (2009)352-358). The anti-drug antibody (ADA) testing usually involves a twotier approach: (1) assays for ADA detection and (2) assays for ADAcharacterization. ADA detection assays include screening and specificityconfirmation (confirmatory) assays. Microtiter plate-based enzyme-linkedimmunosorbent assays (ELISAs) are still the most widely used format toscreen for ADAs due to their high-throughput efficiency, relativesimplicity and high sensitivity (Geng, D., et al., J. Pharm. Biomed.Anal. 39 (2005) 364-375). ADA ELISAs are most often designed in a bridgeformat which provides high selectivity, detection of all isotypes andpan-species ADA detection capability (Mire-Sluis, A. R., et al., J.Immunol. Methods 289 (2004) 1-16).

A bridging ELSA has been developed and used as a screening andconfirmation ADA assay for the anti-IL6R antibody tocilizumab(Stubenrauch, K., et al., Clin. Ther. 32 (2010) 1597-1609).

Stubenrauch, K., et al. report a generic anti-drug antibody assay withdrug tolerance in serum samples from mice exposed to human antibodies(Anal. Biochem. 430 (2012) 193-199). Bourdage, J. S., et al. report theeffect of double antigen bridging immunoassay format on antigen coatingconcentration dependence and implications for designing immunogenicityassays for monoclonal antibodies (J. Pharm. Biochem. Anal. 39 (2005)685-690). Mikulskis, A., et al. report solution ELISA as a platform ofchoice for development of robust, drug tolerant immunogenicity assays insupport of drug development (J. Immunol. Meth. 365 (2010) 38-49). Pan,J., et al. report the comparison of the NIDSA® rapid assay with ELISAmethods in immunogenicity testing of two biotherapeutics (J. Pharm. Tox.Meth. 63 (2010) 150-159.

Qiu, Z. J., et al. report a novel homogeneous biotin-digoxigenin basedassay for the detection of human anti-therapeutic antibodies inautoimmune serum (J. Immunol. Meth. 362 (2010) 101-111).

SUMMARY OF THE INVENTION

Herein is reported a bridging enzyme linked immunosorbent assay(bridging ELISA) that can be used as screening, confirmation andfollow-up assay for the detection of anti-drug antibodies (ADA) in serumcontaining samples of patients treated with a therapeutic antibody. Theassay as reported herein is especially useful if the serum containingsample is from a patient with an autoimmune diseases such as rheumatoidarthritis (RA).

The assay as reported herein shows an improved tolerance with respect tothe amount of therapeutic antibody in the sample to be analyzed(increased drug tolerance of the ADA ELISA) and at the same time thenumber of false positive assay results is reduced.

It has been found that with the assay as reported herein interferencesby free drug and by rheumatoid factors (RF) can be minimized.

This assay is especially useful if the sample contains antibodies otherthan the anti-drug antibody in question which can interfere inimmunoassays for the detection of anti-drug antibodies and, thus, wouldaccount for a false positive immunoassay result.

In one embodiment the methods as reported herein are used for thedetermination of anti-drug antibodies of drug antibodies used for ananti-inflammatory therapy.

The increased drug tolerance was achieved by a synergistic interactionof 1) increasing the concentration of biotinylated and digoxigenylatedcapture and tracer reagents; 2) simultaneous, instead of sequentialincubation of the serum sample with the capture and tracer reagents; 3)a prolonged incubation time; 4) use of homogenously mono-coupled captureand tracer reagents; and 5) use of an increased serum matrix content.

The interference from rheumatoid factors can be suppressed by additionof oligomeric human immunoglobulin G (IgG) as an additive.

The drug tolerance of the interference-suppressed ADA assay as reportedherein is at least 10-fold higher than that of assays known in the art.

One aspect as reported herein is an enzyme linked immunosorbent assayfor the detection of anti-drug antibodies against a drug antibody in asample comprising a capture drug antibody and a tracer drug antibody,wherein

-   -   a) the capture drug antibody and the tracer drug antibody are        employed in a concentration of more than 0.5 μg/ml,    -   b) the sample is incubated simultaneously with the capture drug        antibody and the tracer drug antibody for 4 to 24 hours,    -   c) the capture drug antibody and the tracer drug antibody are        derivatized via a single lysine residue,    -   d) the sample comprises 7.5% serum or more, and    -   e) oligomeric human IgG is added to the sample prior to the        incubation with the capture drug antibody and the tracer drug        antibody.

One aspect as reported herein is an enzyme linked immunosorbent assayfor the detection of anti-drug antibodies against a drug antibody in asample of a rheumatoid arthritis patient comprising a capture drugantibody and a tracer drug antibody, wherein

-   -   a) the capture drug antibody and the tracer drug antibody have a        concentration of 0.5 μg/ml or more in the enzyme linked        immunosorbent assay,    -   b) the sample is incubated simultaneously with the capture drug        antibody and the tracer drug antibody for 0.5 to 24 hours,    -   c) the capture drug antibody is a 1:1 conjugate of the capture        drug antibody and a first component of a specific binding pair        and the tracer drug antibody is a 1:1 conjugate of the tracer        drug antibody and a detectable label,    -   d) the sample comprises 1% serum or more, and    -   e) oligomeric human IgG is added to the sample prior to the        incubation with the capture drug antibody and the tracer drug        antibody.

In one embodiment the sample comprises 5% serum or more. In oneembodiment the sample comprises 7.5% serum or more.

In one embodiment the sample comprises anti-drug antibodies andrheumatoid factors.

In one embodiment the drug antibody is an antibody for the treatment ofan inflammatory disease. In one embodiment the antibody for thetreatment of an inflammatory disease is an antibody for the treatment ofan autoimmune disease. In one embodiment the autoimmune disease isrheumatoid arthritis or juvenile arthritis or osteoarthritis orCastleman's disease.

In one embodiment the drug antibody is an antibody for the treatment ofcancer. In one embodiment the antibody is for the treatment of myelomaor plasmacytoma.

In one embodiment the drug antibody is an antibody against the IL-6receptor (anti-IL6R antibody), or against the IGF-1 receptor (anti-IGF1Rantibody), or the IL-13 receptor 1 alpha (anti-IL13R1 alpha antibody),or against Ox4OL (anti-Ox40L antibody), or against tumor necrosis factoralpha (anti-TNFalpha antibody). In one embodiment the drug antibody isan anti-IL6R antibody. In one embodiment the anti-IL6R antibody istocilizumab.

In one embodiment the capture drug antibody is conjugated to a solidphase. In one embodiment the conjugation of the capture drug antibody tothe solid phase is performed via a specific binding pair. In oneembodiment the specific binding pair (first component/second component)is selected from Streptavidin or Avidin/biotin, or antibody/antigen(see, for example, Hermanson, G. T., et al., Bioconjugate Techniques,Academic Press, 1996), or lectin/polysaccharide, or steroid/steroidbinding protein, or hormone/hormone receptor, or enzyme/substrate, orIgG/Protein A and/or G.

In one embodiment the capture drug antibody is conjugated to biotin (asfirst component of a specific binding pair). In this case theconjugation to the solid phase is performed via immobilized Avidin orStreptavidin.

In one embodiment the tracer drug antibody is conjugated to a detectablelabel. In one embodiment the tracer drug antibody is conjugated to thedetectable label via a specific binding pair. In one embodiment thespecific binding pair (first component/second component) is selectedfrom Streptavidin or Avidin/biotin, or antibody/antigen (see, forexample, Hermanson, G. T., et al., Bioconjugate Techniques, AcademicPress, 1996), or lectin/polysaccharide, or steroid/steroid bindingprotein, or hormone/hormone receptor, or enzyme/substrate, orIgG/Protein A and/or G.

In one embodiment the tracer drug antibody is conjugated to digoxigenin(as detectable label). In this case linking to the detectable label isperformed via an antibody against digoxigenin.

In one embodiment the capture drug antibody and the tracer drug antibodyhave a concentration in the enzyme linked immunosorbent assay (ELISA) ofabout 0.5 μg/ml to about 10 μg/ml. In one embodiment the capture drugantibody and the tracer drug antibody have a concentration of more than0.5 μg/ml to less than 10 μg/ml. In one embodiment the capture drugantibody and the tracer drug antibody have a concentration of about 1μg/ml to about 5 μg/ml. In one embodiment the capture drug antibody andthe tracer drug antibody have a concentration of about 1.4 μg/ml toabout 1.8 μg/ml. In one embodiment the capture drug antibody and thetracer drug antibody have a concentration of about 1.45 μg/ml to about1.6 μg/ml. In one preferred embodiment the capture drug antibody and thetracer drug antibody have a concentration of about 1.5 μg/ml.

In one embodiment the incubation time is at least 6 hours. In oneembodiment the incubation time is at least 12 hours. In one embodimentthe incubation time is at least 16 hours.

In one embodiment the incubation time is at most 24 hours.

In one embodiment the incubation time is between 4 hours and 24 hours.In one embodiment the incubation time is between 6 hours and 24 hours.In one embodiment the incubation time is between 12 hours and 24 hours.In one preferred embodiment the incubation time is between 12 hours and20 hours. In one embodiment the incubation time is between 14 hours and18 hours. In one embodiment the incubation time is about 16 hours.

In one embodiment the sample comprises 1% to 20% serum. In oneembodiment the sample comprises about 10% serum.

In one embodiment the oligomeric human IgG is added to a finalconcentration of 10 μg/mL to 1000 μg/mL. In one embodiment theoligomeric human IgG is added to a final concentration of 15 μg/mL to500 μg/mL. In one embodiment the oligomeric human IgG is added to afinal concentration of 20 μg/mL to 250 μg/mL. In one embodiment theoligomeric human IgG is added to a final concentration of 25 μg/mL to100 μg/mL. In one preferred embodiment the oligomeric human IgG is addedto a final concentration of about 50 μg/mL.

One aspect as reported herein is an enzyme linked immunosorbent assayfor the detection of anti-drug antibodies against a drug antibody in asample of a rheumatoid arthritis patient comprising a capture drugantibody and a tracer drug antibody, wherein

-   -   a) the capture drug antibody and the tracer drug antibody have a        concentration of about 1.5 μg/ml in the enzyme linked        immunosorbent assay,    -   b) the sample is incubated simultaneously with the capture drug        antibody and the tracer drug antibody for 14 to 16 hours,    -   c) the capture drug antibody is a 1:1 conjugate of the capture        drug antibody and biotin via a lysine residue of the capture        drug antibody and the tracer drug antibody is a 1:1 conjugate of        the tracer drug antibody and digoxigenin via a lysine residue of        the tracer drug antibody,    -   d) the sample comprises 1% to 20% serum, and    -   e) oligomeric human IgG is added to the sample to a final        concentration of 25 μg/mL to 100 μg/mL prior to the incubation        with the capture drug antibody and the tracer drug antibody.

One aspect as reported herein is the use of oligomeric human IgG for thecapture of rheumatoid factors in an anti-drug antibody ELISA.

One aspect as reported herein is a method of treating an individualhaving a disease comprising administering to the individual an effectiveamount of a therapeutic antibody (drug) and determining the presence ofanti-drug antibodies with an assay as reported herein.

One aspect as reported herein is a method of treating an individualhaving an inflammatory disease comprising administering to theindividual an effective amount of an anti-IL6R antibody (drug) anddetermining the presence of anti-anti-IL6R antibody antibodies(anti-drug antibodies) with an assay as reported herein.

In one embodiment the inflammatory disease is an autoimmune disease. Inone embodiment the autoimmune disease is selected from rheumatoidarthritis, juvenile arthritis, osteoarthritis, or Castleman's disease.

In one embodiment the inflammatory disease is mesangial proliferativeglomerulonephritis.

One aspect as reported herein is a method of treating an individualhaving plasmacytoma comprising administering to the individual aneffective amount of an anti-IL6R antibody (drug) and determining thepresence of anti-anti-IL6R antibody antibodies (anti-drug antibodies)with an assay as reported herein.

One aspect as reported herein is a method of treating an individualhaving myeloma comprising administering to the individual an effectiveamount of an anti-IL6R antibody (drug) and determining the presence ofanti-anti-IL6R antibody antibodies (anti-drug antibodies) with an assayas reported herein.

One aspect as reported herein is a method of inhibiting IL6R activity inan individual comprising administering to the individual an effectiveamount of an anti-IL6R antibody to inhibit IL6R activity and determiningthe presence of anti-anti-IL6R antibody antibodies (anti-drugantibodies) with an assay as reported herein.

SUMMARY OF THE FIGURES

FIG. 1 Assay principle of the drug-tolerant anti-drug antibody assayexemplified for anti-IL6R antibody tocilizumab.

FIG. 2 Assay principle of the interference-suppressed anti-drug antibodyassay for anti-IL6R antibody tocilizumab.

FIG. 3 Calibration curve obtained for the interference-suppressedanti-drug antibody assay for anti-IL6R antibody tocilizumab.

FIG. 4 Comparison of drug tolerance in the two-step anti-drug antibodyELISA and in the interference-suppressed anti-drug antibody ELISA asreported herein for the anti-IL6R antibody tocilizumab; signal of 300ng/mL anti-drug antibody in presence of increasing amounts of drug areshown; dotted line: CP conventional assay; solid line: CPinterference-suppressed assay as reported herein; dotted line withcircles: conventional assay; solid line with squares:interference-suppressed assay as reported herein.

FIG. 5 Cut point determination with the interference-suppressedanti-drug antibody ELISA.

FIG. 6 Cut point determination with the interference-suppressedanti-drug antibody ELISA with TCZ-Bi(mono) and TCZ-Dig(mono).

FIG. 7 Signal variations using conventional ELISA in 77 different serumsamples from TCZ-treated RA patients.

FIG. 8 Signal variations using the interference suppressed ELISA asreported herein in 77 different serum samples from TCZ-treated RApatients.

DEFINITIONS

The term “1:1 conjugate” denotes a conjugate consisting of exactly twoentities joined/conjugated to each other via a single covalent bond. Forexample the term “1:1 conjugate of the capture drug antibody and a firstcomponent of a specific binding pair” denotes a chemical conjugateconsisting of exactly one molecule of the capture drug antibodycovalently conjugated via a single chemical bond to exactly one moleculeof the first component of a specific binding pair. Likewise the term“1:1 conjugate of the tracer drug antibody and a detectable label”denotes a chemical conjugate consisting of exactly one molecule of thetracer drug antibody covalently conjugated via a single chemical bond toexactly one detectable label molecule.

The term “drug antibody” according to the invention denotes an antibodywhich can be administered to an individual, so that a sample of saidindividual is suspected to comprise said drug antibody afteradministration. A drug antibody is an antibody that is intended to beadministered to a human for a therapeutic purpose. Within one assay asreported herein the drug antibody, the capture drug antibody and thetracer drug antibody comprise the “same” antibody molecule, e.g.recombinantly produced with the same expression vector and comprisingthe same amino acid sequence. Drug antibodies (therapeutic monoclonalantibodies) are being used widely for the treatment of various diseasessuch as oncological diseases (e.g. hematological and solid malignanciesincluding non-Hodgkin's lymphoma, breast cancer, and colorectal cancer)or inflammatory diseases. Such antibodies are reported, for example, byLevene, A. P., et al., Journal of the Royal

Society of Medicine 98 (2005) 145-152; Groner, B., et al., Curr. Mol.Meth. 4 (2004) 539-547; and Harris, M., Lancet Oncol. 5 (2004) 292-302.

In one embodiment the drug antibody is an antibody which is useful forthe treatment of an inflammatory disease, i.e. an anti-inflammatoryantibody, such as an anti-IL-6 receptor antibody, or an anti-IGF-1receptor antibody, or an anti-IL-13 receptor 1 alpha antibody.

An example (preferably monoclonal) drug antibody is an antibody againstthe IL-6 receptor (anti IL6R antibody). Such an antibody is, forexample, reported by Mihara, et al., Clin. Immunol. 98 (2001) 319-326;Nishimoto, N., et al, Blood 106 (2005) 2627-2632, in clinical trialNCT00046774, or in WO 2004/096274.

An example (preferably monoclonal) drug antibody is an antibody againstthe IGF-1 receptor (anti IGF1R antibody). Such an antibody is, forexample, reported in WO 2004/087756 or in WO 2005/005635.

An example (preferably monoclonal) drug antibody is an antibody againstthe IL-13 receptor alpha (anti IL13Rlalpha antibody). Antibodies againstIL-13R1alpha are known from, e.g., WO 96/29417, WO 97/15663, WO03/080675, Graber, P., et al., Eur. J. Immunol. 28 (1998) 4286-4298;Poudrier, J., et al., J. Immunol. 163 (1999) 1153-1161; Poudrier, J., etal., Eur. J. Immunol. 30 (2000) 3157-3164; Aikawa, M., et al., Cytokine13 (2001) 75-84, and are commercially available from, e.g., R&D SystemsInc. USA. Further exemplary antibodies against IL-13R1alpha are reportedin WO 2006/072564.

The term “drug antibody used for an anti-inflammatory therapy” as usedherein denotes a drug antibody that is directed against a cell surfacereceptor that mediates inflammation. Such receptors are for example theIL-6 receptor, or the IGF-1 receptor, or the IL-13a receptor 1. If asample from a patient, which is treated with such an anti-inflammatorydrug antibody, is analyzed, it has to be determined, whether thepositive result of the method is based on a true anti-drug antibody(true positive result) or on an antibody other than an anti-drugantibody of the sample (false positive result). An example of such acase is a sample from a patient, who has an autoimmune disease such asrheumatism, and, thus, a sample obtained from said patient contains socalled “rheumatoid factors”. The term “rheumatoid factors” as usedherein denotes antibodies binding to human IgG, to be more precisely tothe Fc-region of human IgG. In most cases these “rheumatic factors” areoligomeric binding molecules.

The term “anti-drug antibody” as used herein denotes an antibody, whichis directed against, i.e. binds to, an antigenic region of a drugantibody. This antigenic region may be the variable region, a CDR, theconstant region, or the glycostructure of the drug antibody. In oneembodiment the anti-drug antibody is directed against a CDR of the drugantibody or a secondary modification of the drug antibody resulting fromthe recombinant production of the drug antibody in recombinant cells,such as, CHO cells, HEK cells, Sp2/0 cells, or BHK cells. Generallyanti-drug antibodies are directed against an antigenic region of a drugantibody that is recognized by the immune system of an animal to whichthe drug antibody is administered. The above described antibodies aretermed “specific anti-drug antibodies”.

Drug antibodies are designed to comprise as few as possible antigenicregions. For example, drug antibodies intended for the use in humans arehumanized prior to the application to a human patient in order tominimize the generation of an immune response against the drug antibody.This immune response would be in the form of anti-drug antibodies(ADAs), which are directed against the non-human parts of such ahumanized drug antibodies, such as e.g. the complementary determiningregions in the variable domains (see e.g. Pan, Y., et al., FASEB J. 9(1995) 43-49).

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, Md. (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat.

Antibodies contain a number of reactive moieties, such as, for example,amino groups (lysins, alpha-amino groups), thiol groups (cystins,cysteine, and methionine), carboxylic acid groups (aspartic acid,glutamic acid) and sugar-alcoholic groups. These can be employed forcoupling to a binding partner like a surface, a protein, a polymer (suchas e.g. PEG, Cellulose or Polystyrol), an enzyme, or a member of abinding pair (see e.g. Aslam M. and Dent, A., Bioconjuation MacMillanRef. Ltd. (1999) 50-100).

The term “anti-idiotypic antibody” denotes an antibody, whichspecifically binds to a binding specificity, such as e.g. a bindingsite, of a parent antibody, i.e. an anti-idiotypic antibody is directede.g. against an antigen binding site of a parent antibody.

In one embodiment the anti-idiotypic antibody specifically binds to oneor more of the CDRs of the parent antibody.

In one embodiment the parent antibody is a therapeutic antibody. In oneembodiment the parent antibody is a multispecific antibody. In oneembodiment the parent antibody is a bispecific antibody.

One of the most common reactive groups of proteins is the aliphaticε-amine of the amino acid lysine. In general, nearly all antibodiescontain abundant lysine residues. Lysine amines/amino groups arereasonably good nucleophiles above pH 8.0 (pK_(a)=9.18) and thereforereact easily and cleanly with a variety of reagents to form stablebonds.

Another common reactive group in antibodies is the thiol residue fromthe sulfur-containing amino acid cystine and its reduction productcysteine (or half cystine). Cysteine contains a free thiol group, whichis more nucleophilic than amines and is generally the most reactivefunctional group in a protein. Thiols are generally reactive at neutralpH, and therefore can be coupled to other molecules selectively in thepresence of amines. Since free sulfhydryl groups are relativelyreactive, proteins with these groups often exist with them in theiroxidized form as disulfide groups or disulfide bonds.

In addition to cystine and cysteine, some proteins also have the aminoacid methionine, which is containing sulfur in a thioether linkage. Theliterature reports the use of several thiolating crosslinking reagentssuch as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA), or sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate (Sulfo-LC-SPDP) to provide efficient ways ofintroducing multiple sulfhydryl groups via reactive amino groups.

Reactive esters, particularly N-hydroxysuccinimide (NHS) esters, areamong the most commonly employed reagents for modification of aminegroups. The optimum pH for reaction in an aqueous environment is pH 8.0to 9.0.

Isothiocyanates are amine-modification reagents and form thiourea bondswith proteins. They react with protein amines in aqueous solution(optimally at pH 9.0 to 9.5).

Aldehydes react under mild aqueous conditions with aliphatic andaromatic amines, hydrazines, and hydrazides to form an imineintermediate (Schiffs base). A Schiffs base can be selectively reducedwith mild or strong reducing agents (such as sodium borohydride orsodium cyanoborohydride) to derive a stable alkyl amine bond.

Other reagents that have been used to modify amines are acid anhydrides.For example, diethylenetriaminepentaacetic anhydride (DTPA) is abifunctional chelating agent that contains two amine-reactive anhydridegroups. It can react with N-terminal and ε-amine groups of proteins toform amide linkages. The anhydride rings open to create multivalent,metal-chelating arms able to bind tightly to metals in a coordinationcomplex.

The term “sample” includes, but is not limited to, any quantity of asubstance from a living thing or formerly living thing. Such livingthings include, but are not limited to, humans, mice, monkeys, rats,rabbits, and other animals. In one embodiment the sample is obtainedfrom a monkey, especially a cynomolgus monkey, or a rabbit, or a mouseor rat. Such substances include, but are not limited to, in oneembodiment whole blood, serum, or plasma from an individual, which arethe most widely used sources of sample in clinical routine.

The term “solid phase” denotes a non-fluid substance, and includesparticles (including microparticles and beads) made from materials suchas polymer, metal (paramagnetic, ferromagnetic particles), glass, andceramic; gel substances such as silica, alumina, and polymer gels;capillaries, which may be made of polymer, metal, glass, and/or ceramic;zeolites and other porous substances; electrodes; microtiter plates;solid strips; and cuvettes, tubes or other spectrometer samplecontainers. A solid phase component is distinguished from inert solidsurfaces in that a “solid phase” contains at least one moiety on itssurface, which is intended to interact with a substance in a sample. Asolid phase may be a stationary component, such as a tube, strip,cuvette or microtiter plate, or may be non-stationary components, suchas beads and microparticles. A variety of microparticles that allow bothnon-covalent or covalent attachment of proteins and other substances canbe used. Such particles include polymer particles such as polystyreneand poly (methylmethacrylate); gold particles such as gold nanoparticlesand gold colloids; and ceramic particles such as silica, glass, andmetal oxide particles. See for example Martin, C. R., et al., AnalyticalChemistry-News & Features, 70 (1998) 322A-327A, or Butler, J. E.,Methods 22 (2000) 4-23.

From chromogens (fluorescent or luminescent groups and dyes), enzymes,NMR-active groups, metal particles, or haptens, such as digoxigenin, thedetectable label is selected in one embodiment. In one embodiment thedetectable label is digoxigenin. The detectable label can also be aphotoactivatable crosslinking group, e.g. an azido or an azirine group.Metal chelates which can be detected by electrochemiluminescense arealso in one embodiment signal-emitting groups, with particularpreference being given to ruthenium chelates, e.g. a ruthenium(bispyridyl)₃ ²⁺ chelate. Suitable ruthenium labeling groups aredescribed, for example, in EP 0 580 979, WO 90/05301, WO 90/11511, andWO 92/14138.

The principles of different immunoassays are described, for example, byHage, D. S. (Anal. Chem. 71 (1999) 294R-304R). Lu, B., et al. (Analyst121 (1996) 29R-32R) report the orientated immobilization of antibodiesfor the use in immunoassays. Avidin-biotin-mediated immunoassays arereported, for example, by Wilchek, M., and Bayer, E. A., in MethodsEnzymol. 184 (1990) 467-469.

DETAILED DESCRIPTION OF THE INVENTION

Herein reported is an interference-suppressed anti-drug antibody assayusing serum samples with increased tolerance to free therapeuticantibody and increased resistance to rheumatoid factor interference.

The principle of an anti-drug antibody assay is the capture of anti-drugantibodies (ADAs) in a complex with digoxigenylated drug (drug-Dig) andbiotinylated drug (drug-Bi) (e.g. tocilizumab (TCZ-Dig and TCZ-Bi,respectively)), the latter one leading to immobilization onto astreptavidin-coated plate (SA-MTP). The ADA/drug-Dig complex bound todrug-Bi on the SA-MTP is detected by an anti-digoxigenin antibodyhorseradish peroxidase enzyme conjugate (anti-Dig-HRP). The horseradishperoxidase (HRP) catalyzes a color reaction of the substrate ABTS. Thecolor intensity is proportional to the concentration of the analyte. Thegeneral principle of an anti-drug antibody assay is shown in FIG. 1.

It has been found that without alteration of the general assay principlethe drug and rheumatoid factor tolerance of a conventional anti-drugantibody assay could be increased by

-   -   1) increasing the concentration of biotinylated and        digoxigenylated capture and tracer reagents;    -   2) simultaneous, instead of sequential incubation of the serum        sample with the capture and tracer reagents;    -   3) prolonged incubation of the serum sample with the capture and        tracer reagents;    -   4) use of homogenous capture and tracer reagents instead of a        heterogeneously coupled mixture;    -   5) use of an increased serum matrix;    -   6) inclusion of oligomeric IgG as assay additive; and    -   7) use of mono biotinylated capture and mono digoxigenylated        tracer antibody.

These measures provided for a synergistic effect.

The above measures lead to an interference-suppressed drug-tolerantanti-drug antibody assay for the detection of anti-drug antibodiesagainst a therapeutic drug antibody.

The general principle of the interference-suppressed drug-tolerantanti-drug antibody assay as reported herein is shown in FIG. 2exemplified for the anti-IL6R antibody tocilizumab.

With the assay setup as reported herein drug tolerance of the ADA assaywas increased at least 10-fold in serum samples from patients comparedto a conventional anti-drug antibody assay. At the same timesusceptibility to rheumatoid factors leading to false-positive assayresults was also decreased.

The therapeutic anti-inflammatory antibody tocilizumab (TCZ) is arecombinant humanized monoclonal antibody directed against theinterleukin-6 receptor. It has been shown to be effective in clinicalstudies of rheumatoid arthritis (Ohsugi, Y. and Kishimoto, T., ExpertOpin. Biol. Ther. 8 (2008) 669-681). The ADA screening and confirmationassay used in these studies shows sufficient drug tolerance for typicalTCZ serum concentrations reached at steady state using an intravenousdosing regimen.

But different routes of administration, such as subcutaneousadministration, more frequent administration, and new indications inchildren might result in higher TCZ serum concentrations at steadystate.

Additionally, for example, rheumatoid factors (RF) are oftensignificantly increased in patients with autoimmune diseases, such ase.g. rheumatoid arthritis patients. RFs demonstrate a preferentialbinding to aggregated gamma globulins and are involved in the clearancemechanism of immune complexes in vivo (Tatarewicz, S., et al., J.Immunol. Methods. 357 (2010) 10-16). RFs are preferentially of thepentameric immunoglobulin M (IgM) isotype (Artandi, S. E., et al., Proc.Natl. Acad. Sci. USA 89 (1991) 94-98) and can non-specifically bind withmultivalency and medium affinity to the constant part of the therapeuticantibody leading to a false positive result in an ADA assay. Forexample, affinity purified rabbit anti-human IgM antibody was includedin the sample diluent to overcome the cross reactive IgM antibodyinterference in RA samples (see e.g. Araujo, J., et al., J. Pharm.Biomed. Anal., 55 (2011) 1041-1049).

It has been found that unspecific binding of RF present in the serumsample to the therapeutic antibody could be prevented by addingoligomeric human IgG to the sample prior to performing the ADA assay.The added oligomeric IgG provides for additional targets for the RF andat most eliminates the interference of RF in the ADA assay as reportedherein.

In the following the interference-suppressed ADA assay as reportedherein is exemplified by the analysis of serum samples of tocilizumab(TCZ) treated rheumatoid arthritis patients.

Measures to increase drug tolerance of the conventional anti-drugantibody assay for detecting anti-drug antibodies against the anti-IL6Rantibody tocilizumab were

-   -   1) increasing the concentration of biotinylated and        digoxigenylated TCZ (e.g. from 0.5 μg/mL to 1.5 μg/mL);    -   2) simultaneous incubation of the serum sample with TCZ-Bi and        TCZ-Dig;    -   3) prolonged incubation of the serum sample with TCZ-Bi and        TCZ-Dig (e.g. from 1 hour to 16 hours);    -   4) use of only lysine-coupled TCZ-Bi and TCZ-Dig reagents        instead of a mixture of lysine- and carbohydrate-coupled        reagents;    -   5) use of an increased serum matrix content; and    -   6) addition of oligomeric human IgG to the sample prior to        incubation TCZ-Bi and TCZ-Dig.

Drug Tolerance

Concentrations of the anti-IL6R antibody tocilizumab to be detected inclinical samples are 0.5 μg/mL or higher, often in the range of from 1μg/mL to 10 μg/mL.

The drug tolerance of the conventional anti-drug antibody assay wasevaluated by determining the highest TCZ concentration at which a givenconcentration of positive control ADA can be detected above thecut-point. Table 1 presents a summary of the results.

TABLE 1 Determination of drug (tocilizumab) tolerance in theconventional anti- drug antibody ELISA. Left-aligned signal values arebelow the plate- specific cut-point of 0.136 AU. tocilizumab [μg/mL] ADA6250 1250 250 50 10 0 [ng/mL] signal mean [AU] 100000 0.049 0.109 0.9493.094 3.169 3.144 10000 0.050 0.062 0.156 1.265 3.117 3.387 1000 0.0520.058 0.071 0.189 0.865 2.141 500.0 0.051 0.057 0.065 0.120 0.448 1.282250.0 0.055 0.060 0.065 0.092 0.255 0.703 125.0 0.056 0.060 0.064 0.0760.157 0.385 62.5 0.056 0.060 0.062 0.067 0.108 0.226 0 0.059 0.061 0.0620.062 0.060 0.071

An ADA concentration of 125 ng/mL was detected and tested positive inthe presence of 10 μg/mL TCZ.

The drug tolerance of the interference-suppressed drug-tolerantanti-drug antibody assay as reported herein was evaluated by determiningthe highest TCZ concentration at which a given concentration of positivecontrol ADA can be detected above the cut-point. Table 2 presents asummary of the results.

TABLE 2 Determination of drug (tocilizumab) tolerance in theinterference- suppressed drug-tolerant anti-drug antibody ELISA asreported herein. Left-aligned signal values are below the plate-specificcut-point of 0.045 AU. tocilizumab [μg/mL] ADA 100 30 10 3 1 0 [ng/mL]signal mean [AU] 10,000 0.948 2.263 3.266 3.536 3.146 3.272 3,000 0.3100.943 1.956 >3.5 2.860 3.161 1,000 0.119 0.364 0.789 1.051 1.180 2.292300 0.055 0.133 0.280 0.376 0.426 0.857 100 0.033 0.057 0.105 0.1440.163 0.310 30 0.027 0.036 0.051 0.063 0.068 0.113 10 0.022 0.025 0.0320.039 0.040 0.056 0 0.024 0.023 0.026 0.027 0.027 0.027

In the interference-suppressed drug-tolerant anti-drug antibody ELISA asreported herein a very low ADA concentration of 30 ng/mL was detectedand tested positive in the presence of 10 μg/mL TCZ. Furthermore ADAconcentrations of 100 ng/mL and 300 ng/mL show a drug antibody toleranceof 30 μg/ml and even 100 μg/mL, respectively.

In comparison with the same experiments conducted with the previouslyused, two-step conventional anti-drug antibody ELISA for tocilizumabrevealed an at least 10-fold higher drug tolerance with theinterference-suppressed assay (see also FIG. 4 for 300 ng/mL ADAconcentration).

The drug tolerance of the interference-suppressed drug-tolerantanti-drug antibody assay as reported herein with 1:1 conjugates of themonovalent bonded biotin and digoxygenin to the capture and tracer drugantibody, respectively, was evaluated by determining the highest TCZconcentration at which a given concentration of positive control ADA canbe detected above the cut-point. Table 3 presents a summary of theresults.

TABLE 3 Determination of drug (tocilizumab) tolerance in theinterference- suppressed drug-tolerant anti-drug antibody ELISA with 1:1conjugates of biotin and digoxygenin to the capture and tracer drugantibody as reported herein. Left-aligned signal values are below theplate-specific cut-point of 0.037 AU. tocilizumab [μg/mL] ADA 80 70 6050 5 0 [ng/mL] signal mean [AU] 500 0.090 0.097 0.112 0.122 0.380 1.071250 0.057 0.061 0.067 0.075 0.215 0.653 125 0.043 0.045 0.048 0.0530.133 0.343 0.0 0.025 0.024 0.026 0.027 0.030 0.027

In the interference-suppressed drug-tolerant anti-drug antibody ELISA asreported herein an ADA concentration of 250 ng/mL was detected andtested positive in the presence of 80 μg/mL TCZ.

Interference Suppression:

Sixteen clinical serum samples were analyzed by a conventional anti-drugantibody assay as described in Stubenrauch et al. (supra). The resultsare summarized in Table 4a.

TABLE 4a Results of the analysis of 16 serum samples from rheumatoidarthritis patients treated with tocilizumab with the ADA assay accordingto Stubenrauch et al. (supra). conventional sample No. ADA assay 1 + 2 +3 + 4 + 5 + 6 + 7 + 8 − 9 − 10 − 11 − 12 − 13 − 14 − 15 − 16 +

Without alteration of the assay principle, a series of measures weretaken to obtain an interference-suppressed drug tolerant anti-drugantibody ELISA as reported herein.

These are:

-   -   1) increasing the concentration of biotinylated and        digoxigenylated TCZ (e.g. from 0.5 μg/mL to 1.5 μg/mL);    -   2) simultaneous incubation of the serum sample with TCZ-Bi and        TCZ-Dig;    -   3) prolonged incubation of the serum sample with TCZ-Bi and        TCZ-Dig (e.g. from 1 hour to 16 hours);    -   4) use of only lysine-coupled TCZ-Bi and TCZ-Dig reagents        instead of a mixture of lysine- and carbohydrate-coupled        reagents;    -   5) use of an increased serum matrix content; and    -   6) addition of oligomeric human IgG to the sample prior to        incubation TCZ-Bi and TCZ-Dig.

The results as obtained with the interference suppressed assay asreported herein is shown in the following Table 4b.

TABLE 4b Comparative analysis of 16 serum samples from rheumatoidarthritis patients treated with tocilizumab with the conventional andthe herein reported interference suppressed ADA assay. interferenceconventional suppressed sample No. ADA assay ADA assay 1 + + 2 + + 3 + +4 + + 5 + + 6 + + 7 + + 8 − − 9 − − 10 − − 11 − − 12 − − 13 − − 14 − −15 − + 16 + −

It has been found that if only a part of the measures as outlined abovewere taken the reduction of interference was not sufficient and still asusceptibility to interference by rheumatoid factors existed resultingin false-positive ADA assay results.

If, for example, only the measures

-   -   1) increasing the concentration of biotinylated and        digoxigenylated TCZ (e.g. from 0.5 μg/mL to 1.5 μg/mL);    -   2) simultaneous incubation of the serum sample with TCZ-Bi and        TCZ-Dig;    -   3) prolonged incubation of the serum sample with TCZ-Bi and        TCZ-Dig (e.g. from 1 hour to 16 hours);    -   4) use of only lysine-coupled TCZ-Bi and TCZ-Dig reagents        instead of a mixture of lysine- and carbohydrate-coupled        reagents; and    -   5) use of an increased serum matrix content        were taken not the full reduction of susceptibility to false        positive assay results can be seen. The comparative data is        shown in Table 4c.

TABLE 4c Comparative analysis of 16 serum samples from rheumatoidarthritis patients treated with tocilizumab with the different formatsof the ADA assay. interference- suppressed ADA assay as reported hereinsample without with No. additive additive 1 + + 2 + + 3 + + 4 + + 5 + +6 + + 7 + + 8 + − 9 − − 10 + − 11 + − 12 + − 13 + − 14 + − 15 + + 16 + −

Comparative evaluation of 258 different serum samples from TCZ-treatedRA patients with the conventional ADA assay and theinterference-suppressed drug-tolerant ADA assay as reported hereinshowed the same positive results in 12 Samples. The conventional assaymeasured 27 placebo patients positive whereas theinterference-suppressed drug-tolerant ADA assay as reported herein only4. In conclusion, the set of measures as described herein and theaddition of oligomeric human IgG as an ADA assay additive conferredincreased drug tolerance and suppressed interference by RF compared tothe conventional ADA assay.

A subset analysis of above-mentioned data is shown in Table 7 below.

TABLE 7 interference- conventional suppressed Patient ELISA ELISA P1 +0.812 + 0.110 P2 + 1.009 + 0.893 P2 − 0.097 + 0.088 P2 + 0.276 + 0.228P3 + 0.349 + 0.566 P3 + 2.405 + 1.760 P3 + 1.307 + 1.001 P4 + 1.409 +0.242 P5 + 0.219 + 0.060 P6 + 0.387 + 0.688 P6 + 3.689 + 3.721 P6 +3.506 + 3.722 P7 + 0.771 + 0.058 study related CP: 0.215 (conventionalELISA); 0.058 (interference-suppressed ELISA); +: positive ELISA result;−: negative ELISA result

In Table 8a assay signals for 27 placebo patients not treated with TCZare shown. Due to the absence of TCZ-treatment induced ADA, high assaysignals in this group are not expected and would indicate a potentialinterference.

TABLE 8a interference- conventional suppressed Patient ELISA ELISA P8 +0.270 − 0.032 P8 + 0.231 − 0.033 P9 + 3.736 − 0.055 P9 + 3.297 + 0.058P9 + 2.863 − 0.046 P9 + 3.739 − 0.052 P1 + 0.812 + 0.110 P10 + 0.755 −0.021 P10 + 0.635 − 0.021 P11 + 0.272 − 0.020 P11 + 0.271 − 0.021 P11 +0.234 − 0.020 P12 + 1.157 − 0.027 P12 + 1.362 − 0.028 P12 + 1.149 −0.029 P4 + 0.522 − 0.033 P4 + 1.409 + 0.242 P4 + 0.651 − 0.047 P13 +0.349 − 0.023 P14 + 0.245 − 0.033 P14 + 0.276 − 0.035 P14 + 0.275 −0.037 P15 + 0.580 − 0.049 P7 + 0.990 − 0.056 P7 + 0.822 − 0.052 P7 +0.523 − 0.042 P7 + 0.771 + 0.058 study related cut-point (CP): 0.215(conventional ELISA); 0.058 (interference-suppressed ELISA); +: positiveELISA result; −: negative ELISA result

Signal pattern of both assay are very different: whereas all 27 placebossamples were determined to be positive using the conventional ELISA, butonly 4 out of the 27 sample were determined to be positive with theinterference-suppressed ELISA as reported herein.

In addition to placebo-treated patients sample of TCZ-treated patientshave been analyzed. In Table 8b results for the patients prior toTCZ-treatment are shown. In Table 8c results for TCZ-treated patientsare shown.

TABLE 8b interference- conventional suppressed Patient Time point ELISAELISA dosing P2 baseline + 1.009 + 0.893 4 P16 baseline + 0.745 − 0.0218 P17 baseline + 0.281 − 0.020 8 P18 baseline + 1.401 − 0.023 4 studyrelated CP: 0.215 (conventional ELISA); 0.058 (interference-suppressedELISA); +: positive ELISA result; −: negative ELISA result

TABLE 8c interference- conventional suppressed Patient Time point ELISAELISA dosing P19 week 24 + 0.281 − 0.030 4 mg/kg P19 week 24 + 0.226 −0.031 4 mg/kg P19 week 4 + 0.293 − 0.030 4 mg/kg P19 week 8 + 0.362 −0.028 4 mg/kg P2 week 4 − 0.097 + 0.088 4 mg/kg P2 week 8 + 0.276 +0.228 4 mg/kg P16 week 24 + 0.416 − 0.030 8 mg/kg P16 week 4 + 0.542 −0.029 8 mg/kg P16 week4 + 0.397 − 0.024 8 mg/kg P3 week 24 + 2.405 +1.760 4 mg/kg P3 week 28 + 1.307 + 1.001 4 mg/kg P3 week 4 − 0.181 +0.120 4 mg/kg P3 week 8 + 0.349 + 0.566 4 mg/kg P20 week 8 + 0.818 −0.034 4 mg/kg P21 week 4 + 0.289 − 0.031 8 mg/kg P17 week 12 + 0.369 −0.023 8 mg/kg P17 week 24 + 0.330 − 0.022 8 mg/kg P17 week 4 + 0.488 −0.022 8 mg/kg P17 week 4 + 0.465 − 0.025 8 mg/kg P17 week 8 + 0.409 −0.026 8 mg/kg P18 week 4 + 1.218 − 0.026 4 mg/kg P18 week 4 + 1.041 −0.028 4 mg/kg P5 week 28 + 0.219 + 0.060 8 mg/kg P6 week 12 + 3.689 +3.721 4 mg/kg P6 week 24 + 3.506 + 3.722 4 mg/kg P6 week 8 + 0.387 +0.688 4 mg/kg P22 week24 + 0.423 − 0.020 4 mg/kg study related CP: 0.215(conventional ELISA); 0.058 (interference-suppressed ELISA); +: positiveELISA result; −: negative ELISA result

The assay as reported herein provides a benefit independent from thetherapeutic antibody and the target employed. This is shown in thefollowing Table for an anti-IL6R antibody, an anti-IGF-1R antibody, ananti-IL13Ralpha antibody, an anti-OX40L antibody and an anti-Abetaantibody using samples of patients being diagnosed positive forrheumatoid arthritis.

TABLE 9a anti-IL6R antibody interference- conventional suppressedPatient ELISA ELISA P23 + 0.169 − 0.079 P24 + 0.197 − 0.082 P25 + 0.240− 0.110 P26 − 0.131 − 0.088 P27 + 0.215 − 0.111 P28 + 0.199 − 0.136 P29− 0.135 − 0.085 P30 + 0.220 − 0.086 P31 + 0.158 − 0.100 P32 + 0.221 −0.132 P33 − 0.110 − 0.081 P34 − 0.099 − 0.090 P35 − 0.100 − 0.082 P36 −0.098 − 0.076 CP 0.157 0.140

TABLE 9b anti-IGF-1R antibody interference- conventional suppressedPatient ELISA ELISA P23 − 0.153 − 0.127 P24 + 0.423 − 0.132 P25 + 0.266− 0.145 P26 − 0.171 − 0.163 P27 − 0.152 − 0.132 P28 − 0.133 − 0.120P29 + 0.245 − 0.124 P30 − 0.173 − 0.142 P31 − 0.152 − 0.115 P32 − 0.172− 0.131 P33 − 0.131 − 0.134 P34 − 0.124 − 0.115 P35 + 0.189 + 0.189 P36− 0.157 − 0.154 CP 0.176 0.185

TABLE 9c anti-IL13Ralpha antibody interference- conventional suppressedPatient ELISA ELISA P23 + 0.556 − 0.080 P24 + 0.881 + 0.198 P25 +2.192 + 0.761 P26 + 0.674 + 0.235 P27 + 0.604 − 0.044 P28 + 0.177 −0.000 P29 − 0.000 − 0.006 P30 + 0.424 − 0.091 P31 + 0.342 − 0.000 P32 +0.353 − 0.092 P33 + 0.208 − 0.021 P34 − 0.000 − 0.000 P35 − 0.079 −0.097 P36 + 0.238 + 0.235 CP 0.100 0.100

TABLE 9d anti-OX40L antibody interference- conventional suppressedPatient ELISA ELISA P23 + 0.148 − 0.073 P24 + 0.477 − 0.066 P25 +0.414 + 0.087 P26 − 0.103 − 0.067 P27 + 0.137 − 0.072 P28 + 0.211 +0.126 P29 − 0.116 − 0.074 P30 + 0.436 − 0.064 P32 + 0.198 − 0.079 P33 +0.122 − 0.078 P34 − 0.096 − 0.070 P35 − 0.088 − 0.067 P36 + 0.121 −0.076 CP 0.117 0.085

TABLE 9e anti-Abeta antibody interference- conventional suppressedPatient ELISA ELISA P25 + 0.063 − 0.025 P26 − 0.025 − 0.026 P28 +0.086 + 0.080 P29 − 0.020 − 0.021 P30 − 0.037 − 0.022 P31 − 0.036 −0.024 P32 − 0.029 − 0.024 P33 − 0.027 − 0.026 P34 + 0.069 + 0.069 P35 −0.019 − 0.021 P36 − 0.033 − 0.032 CP 0.044 0.042

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES Materials and Methods

Purified pooled human immunoglobulin class G (IgG) was prepared asdescribed by Stubenrauch et al. (Anal. Biochem. 390 (2009) 189-196).Briefly, pooled human serum from healthy donors has been delipidatedwith Aerosil (silicon dioxide, 1.5% (w/v)) and precipitated withammonium sulfate (ad 2.0 M). The pellet was homogenized in phosphatebuffer and dialyzed against phosphate buffer, pH 7.0.

The mixture was separated by DEAE ion exchange chromatography at pH 7.0and the IgG in the flow through was concentrated to 5.93 mg/mL andpurified by gel filtration.

Polyclonal anti-digoxigenin-horse radish peroxidase (HRP) conjugate (Fabfragments) was obtained from Roche Diagnostics GmbH, Mannheim, Germany(cat. no. 11633716). Polyclonal rabbit anti-TCZ antibodies (0.5mg-equivalent/mL) used as positive quality controls (QC) and calibrationstandards (CS) were prepared as described in Stubenrauch et al. (supra).

Individual human serum samples were provided by the serum bank of RocheDiagnostics GmbH, Penzberg, Germany. Pooled human serum matrix fornegative control was supplied by TCS Biosciences Ltd., Buckingham, UK.

The following reagents were obtained from Roche Diagnostics GmbH,Mannheim, Germany: 2,2′ -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid(ABTS) substrate (cat. no. 11684302-001), the washing buffer for theELISA: phosphate-buffered saline (PBS) (0.01 M KH2PO4, 0.1 M Na2HPO4,1.37 M NaCl, 0.027 M KCl; pH 7.0)/0.05% polysorbate 20 (Tween 20) (cat.no. 11332465-001) and the ready-to-use Universal buffer (cat no.4742672)which was used as dilution buffer in the ELISA. All chemicals were ofanalytical grade.

Streptavidin-coated microtiter plates (SA-MTP) were obtained fromMicroCoat Biotechnologie GmbH, Bernried. Uncoated Nunc 96-microwellplates were from Fisher Scientific GmbH, Schwerte, Germany (cat no.442587) and used for pre-incubation.

Conventional Anti-Drug Antibody Assay:

The assay was performed at room temperature. In the first step, TCZ-Biwas bound to SA-MTPs at a concentration of 0.5 μg/ml by incubating 100μL on a shaker at 400 rpm for 1 hour. Before adding the pre-incubationsolution to the SA-MTPs, the excess unbound TCZ-Bi was removed bywashing 3 times. In parallel with the coating procedure, pre-incubationof standards and samples was performed in duplicate in a separateuncoated 96-well plate. The samples and standards were diluted (1:10) inthe wells with 10% serum matrix to a volume of 75 μL and mixed with thesame volume of TCZ-DIG, starting the one hour pre-incubation period. TheTCZ-Bi-coated SA-MTPs were loaded with the pre-incubation solutions bytransferring 100 μL from each well of the pre-incubation plate to thewells of the coated MTP and incubated on a shaker at 400 rpm for onehour. After washing, polyclonal anti-Dig horseradish peroxidase (HRP)conjugate in a volume of 100 μL (100 mU/mL) was added to the wells andincubated on a shaker for one hour. After washing, the HRP-catalyzedcolor-generating reaction was initiated by adding 100 μL ABTS solution.When the maximum optical density (OD) was about 2.0, usually within 20to 30 minutes, the signal of the color reaction was measured by an ELISAreader at a wave length of 405 nm (reference, 490 nm). The same assaywas performed in the presence of the confirmation reagents, withsimultaneous measurement without the confirmation reagents. The obtainedOD data were used for generating the standard calibration curve bynonlinear 4-parameter regression curve fitting according to theWiemer-Rodbard method for calculating the sample concentration.

The cutoff points for the test results were set at the 95% CIs of theassay signals (OD) from multiple analyses of human blank serum samplesfrom healthy volunteers and patients with RA. A screening test resultwas considered positive at a value above the cutoff. A decrease inabsorbance of >20% relative to the non-spiked sample indicated apositive result. The screening cutoff was determined at 61.4 ng/mL ofreference antibody. Intra-assay and inter-assay accuracy were 84.8% to93.1% and 91.3% to 92.2%, respectively. The corresponding values forintra-assay and inter-assay precision were 1.8% to 2.0% and 6.8% to8.0%. The accuracy of the ELISA was defined by the extent to which theresults of the assay agreed with the true values. Accuracy wasdetermined by comparing measured concentrations of the rabbit polyclonalanti-TCZ-positive control standard spiked into human serum with nominalconcentrations of anti-TCZ. High-concentration (360 ng equivalents/mL)and low-concentration (60 ng equivalents/mL) positive control standardswere analyzed in six aliquots of each positive control standard measuredin duplicate to determine intra-assay accuracy and in three aliquots ofeach positive control standard measured in duplicate to determineinter-assay accuracy.

Example 1 Biotinylation of Anti-IL6R Antibody Tocilizumab

a) Preparation of Conventionally Biotinylated IgG

The anti-IL6R antibody tocilizumab has been dialyzed against buffer (100mM potassium phosphate buffer (in the following denoted as K-PO4), pH8.5). Afterwards the solution was adjusted to a protein concentration of5 mg/ml. D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester wasdissolved in dimethyl sulphoxide (DMSO) and added to the antibodysolution in a molar ratio of 1:5. After 60 minutes the reaction wasstopped by adding L-lysine. The surplus of the labeling reagent wasremoved by dialysis against 50 mM K-PO4 supplemented with 150 mM KCl, pH7.5. Aliquots of TCZ-Bi were stored including 6.5% sucrose at −80° C.

b) Preparation of Mono Biotinylated IgG

The anti-IL6R antibody tocilizumab has been dialyzed against 100 mMK-PO4, pH 8.5 and afterwards the solution was adjusted to a proteinconcentration of 5 mg/ml. D-biotinoyl-aminocaproicacid-N-hydroxysuccinimide ester was dissolved in dimethyl sulphoxide(DMSO) and added to the antibody solution in a molar ratio of 1:1. After60 minutes the reaction was stopped by adding L-lysine. The surplus ofthe labeling reagent was removed by dialysis against 25 mM K-PO4supplemented with 150 mM KCl, pH 7.2. The mixture was transferred to abuffer with 100 mM K-PO4, 150 mM KCl, pH 7.2 including 1 M ammoniumsulfate and applied to a column with streptavidin mutein sepharose. Thenon biotinylated IgG is in the flow through, the mono biotinylated IgGis eluted with 100 mM K-PO4, 150 mM KCl, 1.5% DMSO, pH 7.2 and thebiotinylated IgG including higher biotinylated populations is elutedwith 100 mM K-PO4, 150 mM KCl, 2 mM D-biotin, pH 7.2.

The mono biotinylated antibody was dialyzed against 50 mM K-PO4supplemented with 150 mM KCl, pH 7.5. The aliquots were stored including6.5% sucrose at −80° C.

Example 2 Digoxigenylation of Anti-IL6R Antibody Tocilizumab

a) Preparation of Conventionally Digoxigenylated IgG

The anti-IL6R antibody tocilizumab has been dialyzed against buffer (100mM potassium phosphate buffer (in the following denoted as K-PO4), pH8.5). Afterwards the solution was adjusted to a protein concentration of5 mg/ml. Digoxigenin 3 -O-methylcarbonyl-ε-aminocaproicacid-N-hydroxysuccinimide ester was dissolved in DMSO and added to theantibody solution in a molar ratio of 1:4. After 60 minutes the reactionhas been stopped by adding L-lysine. The surplus of labeling reagent wasremoved by dialysis against 50 mM K-PO4 supplemented with 150 mM NaCl,pH 7.5. Digoxigenylated TCZ (TCZ-Dig) was stored in aliquots including6.5% sucrose at −80° C.

b) Preparation of Mono Digoxigenylated IgG

The anti-IL6R antibody tocilizumab has been dialyzed against 100 mMK-PO4, pH 8.5 and afterwards the solution was adjusted to a proteinconcentration of 5 mg/ml. Digoxigenin 3-O-methylcarbonyl-ε-aminocaproicacid-N-hydroxysuccinimide ester was dissolved in dimethyl sulphoxide(DMSO) and added to the antibody solution in a molar ratio of 1:1. After60 minutes the reaction was stopped by adding L-lysine. The surplus ofthe labeling reagent was removed by dialysis against 50 mM K-PO4supplemented with 150 mM KCl, pH 7.5. The mixture was applied to asepharose column with immobilized monoclonal antibodies againstdigoxigenin. The non digoxigenylated antibody is in the flow through,the mono digoxigenylated IgG is eluted with gentle elution buffer(Thermo Scientific, # 21013) and the digoxigenylated antibody includinghigher digoxigenylated populations is eluted with 1 M propionic acid.The fraction with mono digoxigenylated antibody was dialyzed firstagainst 20 mM TRIS, 20 mM NaCl, pH 7.5 and second against 50 mM K-PO4,150 mM KCl, pH 7.5. The aliquots were stored including 6.5% sucrose at−80° C.

Example 3 Generation of Human IgG in Oligomeric Form

Human IgG purified from human serum by ion exchange chromatography wasdialyzed against 150 mM potassium phosphate buffer containing 100 mMNaCl, pH 8.4, and the protein solution was concentrated to a proteinconcentration of 50 mg/ml. Disuccinimidyl suberate (DSS) was dissolvedin DMSO and added to the antibody solution in a molar ration of 1:6(IgG:DSS). The mixture was incubated at 25° C. and pH 8.4 with stirringand the reaction was analyzed with an analytical gel filtration column(e.g. using a TSK 4000 column). The polymerization was stopped after 140min. by adding lysine to a final concentration of 20 mM. After 45 min.incubation at 25° C. the oligomeric human IgG was separated by gelfiltration (e.g. using a Sephacryl 5400 column) to remove low molecularfractions. The composition of the oligomers was characterized by UVspectroscopy, size exclusion chromatography and SDS-PAGE gelelectrophoresis. The oligomeric human IgG was aliquoted (10.5 mg/mL) andstored at −65° C. until it was freshly diluted with Universal buffer(cat no. 4742672) to a concentration of 55.6 μg/mL for use as ADA assayadditive (AAA) in the immunoassay.

Example 4 Preparation of Calibration Standards and Quality ControlSamples

Stock solutions for calibration standards (CS) and quality controlsamples (QC) were prepared separately. The CS samples were freshlyprepared at the assay day using a 0.5 mg/mL stock solution of TCZ. Afterpre-dilution with human pooled serum (HPS), the resulting CS workingsolution was stepwise 1:1 diluted with 100% HPS to yield calibratorconcentrations of 1,000; 500; 250; 125; 62.5; 31.3; and 15.6 ng/mLbefore use in the assay. The negative control was 100% HPS. For thepre-incubation step in the assay, the CS samples were diluted 1:10 toadjust to a serum concentration of 10% and an assay concentration rangeof 100 ng/mL to 1.56 ng/mL.

The QC stock samples used were made in 100% human pooled serum andstored as single use aliquots at −20° C. Three separate QC samples wereprepared and stored at stock concentrations representing high (750ng/mL), medium (400 ng/mL) and low (50 ng/mL) undiluted serumconcentrations. For the pre-incubation step in the assay the QC sampleswere freshly diluted 1:10 in the capture/detection solution to reach aserum concentration of 10%. A fourth QC sample at the typical cut pointof the assay at 25 ng/mL was further used.

Example 5 ADA Screening and Confirmation Assay

A sandwich ELISA was used for both screening and confirmation ofanti-drug antibodies (ADAs) against tocilizumab (TCZ) (see Stubenrauch,K., et al., Clin. Ther. 32 (2010) 1597-1609). The principle of themethod is the capture of ADAs in complex with TCZ-Dig and TCZ-Bi, thelatter one leading to immobilization onto a streptavidin-coated plate.The TCZ-Bi/ADA/TCZ-Dig complex bound to the SA-MTP was detected by ananti-Dig-HRP enzyme conjugated antibody. The principle of thedrug-tolerant anti-drug antibody assay is shown in FIG. 1. Inclusion ofoligomeric IgG as ADA assay additive leads to an interference-suppressedanti-drug antibody assay as shown in FIG. 2. The horseradish peroxidase(HRP) of the polyclonal antibody catalyzes a color reaction of thesubstrate ABTS. The color intensity is proportional to the concentrationof the analyte.

The screening assay was performed at room temperature. Reagents andserum samples were diluted with Universal buffer (cat no. 4742672), allwashing steps were performed with the washing buffer (PBS, 0.05%polysorbate 20 (Tween 20) (cat. no. 11332465-001)) three times with 300μL per well. Incubations were performed under shaking on a microtiterplate shaker (MTP shaker) at 500 rpm. Test samples, QC and CS sampleswere incubated overnight (up to 16 hours) with the capture antibodyTCZ-Bi and the detection antibody TCZ-Dig. Each well of thepre-incubation microtiter plate (MTP) was loaded with 225 μL of thecapture/detection solution containing 1.667 μg/mL TCZ-Bi and 1.667 μg/mLTCZ-Dig with oligomeric human IgG ADA assay additive (AAA) andthereafter 25 μL of the respective samples were added. The resultingconcentrations of TCZ-Bi and TCZ-Dig were 1.5 μg/mL each and theoligomeric human IgG had a concentration of 50 μg/mL. The loaded MTP wascovered to prevent evaporation and incubated overnight. Duplicates of100 μL of each well of the pre-incubation plate were transferred to thewells of a streptavidin-coated microtiter plate (SA-MTP) which wascovered and incubated for 1 h.

After washing, the polyclonal anti-Dig Fab-HRP conjugate with aconcentration of 25 mU/mL was added in a volume of 100 μL to each welland incubated for 1 h. After washing, the ABTS ready-to-use solution wasadded in 100 μL aliquots to each well and incubated for about 10 to 15min. while shaking. The signal of the color-generating reaction wasmeasured by an ELISA reader at 405 nm wavelength (reference wavelength:490 nm). Absorbance values of each serum sample were determined intriplicates. The highest standard should reach an optical density (OD)between 1.8 and 2.2 arbitrary units (AU). The obtained OD data was usedfor generating the standard calibration curve by non-linear 4-paramterfit “Wiemer Rodbard” for calculating the sample concentration. A samplewas confirmed as positive to ADAs if the recovery of the concentrationwas less than the specificity cut-point.

Evaluation of the specificity cut-point was performed by analysis of 32individual blank human serum samples of rheumatoid arthritis patients induplicates on one MTP. The cut-point specifies the signal above which asample is defined as potentially positive for the presence of ADAs inthe ADA screening assay. Due to non-normality of the data, anon-parametric approach with a 95% percentile was applied for cut-pointcalculation based on the mean of cut-points on replicate plates.

The experiments conducted to determine the cut-point of the ADA assayfrom duplicate measurements of 32 individual human blank serum samplesof rheumatoid arthritis patients revealed a mean AU of about 0,026 onthree different plates with a standard deviation (SD) of about 0.009.The corresponding coefficient of variation (CV) was 23.8%; 20.0%; and19.0%, respectively. Based on these data sets, a normalization factor ofNF=1.6905 was derived which was used throughout the assay qualificationand applied to plate-specific cut-points (plate specific cut-point[AU]=Signal [AU](negative control)×NF).

For qualification of the screening ADA assay, five independentcalibration curve preparations with seven calibrator samples with anassay concentration range of 1.56 ng/mL to 100 ng/mL and a blank samplewere measured in duplicates on one plate. The intra-assay qualificationruns were performed with five replicates (five separate vials) with eachof the four QC samples measured in duplicates on a single plate.Inter-assay qualification data for all QC samples measured in duplicateswere obtained from seven independent test runs performed by at least twooperators on four different days.

A typical calibration curve of the interference-suppressed ELISA isshown in FIG. 3. The precision of duplicate measurements of samples wasassessed during the qualification experiments and its CV did not exceed15%. Intra-assay and inter-assay precision and accuracy values of theinterference-suppressed ADA ELISA are summarized in Table 5.

TABLE 5 Determination of intra-assay and inter-assay precision andaccuracy of the interference-suppressed ELISA using tocilizumab-specificADAs spiked into human serum. ADA concentration in 100% serum [ng/mL]Intra-assay (n = 5) Inter-assay (n = 7) High Mid Low High Mid Low QC QCQC QC QC QC spiked 750 400 50 750 400 50 (expected concentration) meanof 683 382 50.8 696 402 52.5 measured concentration SD of 15.6 15.8 1.3321.2 13.6 2.8 measured concentration precision 2.28 4.14 2.62 3.04 3.385.33 (% CV) accuracy (% 91.1 95.5 102 92.8 101 105 recovery)

The determined intra-assay precision was <5% for all QCs including thecut-point QC of 25 ng/mL. The determined intra-assay accuracy was in therange of 91.1% to 102% and all cut-point QC samples tested positive. Theinter-assay precision for back-calculated QCs was <6% for all QCs. Thedetermined inter-assay accuracy was 92.8% to 105% for the high, mediumand low QCs. All cut-point QC measurements provided ADA-positivity.

A potential high-dose hook effect was assessed by serial titration (1:2)of a positive control sample within an assay concentration range of25,000 ng/mL to 6.1 ng/mL. Recovery of ADA concentrations within theassay range was between 77.9% and 98.9%. For analysis of potentialmatrix effects, 11 individual normal human serum samples were spiked athigh and low dose QC, i.e. 50 and 750 ng/mL in 100% serum, with positivecontrol ADA and were quantified. In addition, QC samples were alsoanalyzed on the same plate. Recovery of the low and high ADAconcentrations was 111% (range: 104 to 117%) and 111% (range: 107 to117%), indicating that there was no matrix effect in theinterference-suppressed ADA ELISA.

The drug tolerance was evaluated by determining the highest TCZconcentration at which a given concentration of positive control ADA canbe detected above the cut-point. Table 2 presents a summary of acomplete data set.

TABLE 2 Determination of drug (tocilizumab) tolerance in theinterference- suppressed drug-tolerant anti-drug antibody ELISA asreported herein. Left-aligned signal values are above the plate-specificcut-point of 0.045 AU, right-aligned values are below the cut-point. TCZ[μg/mL] ADA 100 30 10 3 1 0 [ng/mL] signal mean [AU] 10,000 0.948 2.2633.266 3.536 3.146 3.272 3,000 0.310 0.943 1.956 >3.5 2.860 3.161 1,0000.119 0.364 0.789 1.051 1.180 2.292 300 0.055 0.133 0.280 0.376 0.4260.857 100 0.033 0.057 0.105 0.144 0.163 0.310 30 0.027 0.036 0.051 0.0630.068 0.113 10 0.022 0.025 0.032 0.039 0.040 0.056 0 0.024 0.023 0.0260.027 0.027 0.027

Concentrations of the anti-IL6R antibody tocilizumab to be detected inclinical samples are 0.5 μg/mL or higher, often in the range of from 1μg/mL to 10 μg/mL. A very low ADA concentration of 30 ng/mL was detectedand tested positive in the presence of 10 μg/mL TCZ. Furthermore ADAconcentrations of 100 ng/mL and 300 ng/mL show a drug antibody toleranceof 30 μg/ml and even 100 μg/mL, respectively. The same experimentsconducted with the previously used, two-step conventional ADA ELISA fortocilizumab revealed an at least 10-fold higher drug tolerance with theinterference-suppressed assay (see FIG. 4 for 300 ng/mL ADAconcentration).

The concentration of TCZ to be used as the excess free drug in theconfirmation assay was based on the data set obtained in the druginterference experiments. To evaluate the TCZ concentration that caninhibit high levels of ADAs in the sample, four different concentrationsof positive control samples (1,000; 500; 250; 83.3 ng/mL in 100% serum)were each incubated with increasing concentrations of TCZ (0; 16; 31;63; 125; 250 μg/mL in 100% serum). The TCZ concentration that inhibitedat least 95% (corresponding to less than 5% signal recovery) of themeasured signal at high concentrations of the positive control ADA wasdetermined to be 25 μg/mL of TCZ.

To reduce the likelihood of false-negatives due to affinity differencesof ADAs in study samples during the later in-study testing phase, atwo-fold higher excess free drug concentration of the determined valuewas used for further evaluation, i.e. 40 μg/mL assay concentrationcorresponding to 400 μg/mL in 100% serum.

The minimal signal inhibition value needed for confirming specific ADAswas determined by pre-incubation of 16 individual blank human serumsamples of rheumatoid arthritis patients with TCZ and analyzed induplicates in one test run. The analysis was performed with and without400 μg/mL of free TCZ. It has been found that addition of free TCZreduced the assay signal by a mean of 14.1% ranging from −11.5% to34.8%, and a SD of 11.2%. Applying a 99.9% confidence interval(mean+3.09 SD) resulted in a minimal signal reduction of 49% for blankserum samples. Based on this calculation, a sample was assessedconfirmation positive if the signal decreased by more than 49% inpresence of excess free drug when compared with that in absence of freedrug. As reference samples, the corresponding samples without excess TCZwere used. To demonstrate the reproducibility of signal inhibition inthe confirmation assay, serum samples with high, medium and low positiveQC concentrations were analyzed three times with and without excess TCZat the predefined concentration.

The interference-suppressed ADA assay for TCZ had a measurement range offrom 1,000 ng/mL to 15.6 ng/mL of ADA calibrator in 100% serum.Intra-assay precision was less than 5% for all quality controls and theintra-assay accuracy was 91.1% to 102%. The inter-assay precision andaccuracy were less than 6% and 92.8% to 105%, respectively.

Qualification of the assay did exclude a hook and matrix effect.

It can be seen that the drug tolerance of the interference-suppressedADA assay was at least 10-fold higher than that of the previous version.

The confirmation assay was performed essentially as described before forthe screening assay except that samples were analyzed in parallelwithout and with excess free drug, i.e. TCZ. The confirmationcapture/detection solution contained the same volume of thecapture/detection solution with additional excess TCZ (44.4 μg/mL) toachieve a final assay concentration of 40 μg/mL TCZ after addition ofthe samples. Calculation of the percent signal inhibition underconfirmatory conditions with excess drug was done using the followingequation:

% signal inhibition=100×(1−([AU]drug-pretreated sample/[AU]untreatedsample)).

Example 6 Application of the Interference-Suppressed ADA Assay toClinical Samples

Measures to increase drug tolerance consisting in 1) increasing theconcentration of biotinylated and digoxigenylated TCZ (e.g. to 1.5μg/mL); 2) simultaneous incubation of the serum sample with TCZ-Bi andTCZ-Dig; 3) prolonged incubation of the serum sample with TCZ-Bi andTCZ-Dig (e.g. overnight); 4) use of only lysine-coupled TCZ-Bi andTCZ-Dig reagents instead of a mixture of lysine- andcarbohydrate-coupled reagents; and use of an increased serum matrix(e.g. 10% instead of 5%) resulted in an increase of ADA positives from12/28 to 25/28.

Sixteen clinical serum samples were analyzed by a set of three differentADA assays: the conventional ADA assay, interference-suppressed ADAassay as reported herein without added oligomeric human IgG, and theinterference-suppressed ADA assay as reported herein with the additionof oligomeric human IgG. The results are summarized in Table 6. Of the16 samples, 15 were tested positive in the version of the ADA assaywherein only measures 1 to 5 have been taken whereas only 8/16 testedpositive in the conventional as well as in the interference-suppresseddrug-tolerant ADA assay as reported herein wherein measures 1 to 6 havebeen taken, with identical results in seven of the eight samples. Theseseven samples with identical results were characterized by low RFconcentrations in the samples and/or at baseline. All seven samplescontained ADAs of the IgG isotype which bound to the Fab part oftocilizumab indicative of true tocilizumab specific ADAs. Three of theseven samples also had IgM isotype ADAs, but which also bound to the Fabpart. In contrast, the vast majority of the remaining samples had ADAspredominantly of the IgM isotype which bound to the constant Fc part oftocilizumab. These samples also contained a high, i.e. >1,000 U/mL,concentration of RF.

TABLE 6 Comparative analysis of 16 serum samples from rheumatoidarthritis patients treated with tocilizumab with the different formatsof the ADA assay, in the BIAcore assay and in the rheumatoid factorassay. interference- BIAcore: suppressed ADA con- isotype/ assay asreported ven- epitope herein tional binding of RF assay [U/ml] samplewithout with ADA IgG IgM study No. additive additive assay ADA ADAsample baseline 1 + + + IgG/ IgG — 312 Fab 2 + + + IgG/ — 324 324 Fab3 + + + IgG/ — 591 324 Fab 4 + + + IgG/ — 56 37 Fab 5 + + + IgG/ IgG/117 129 Fab Fab 6 + + + IgG/ IgG/ — 47 Fab Fab 7 + + + IgG/ — — <15 Fab8 + − − IgG/ IgG/ 2,870 1,305 Fc Fc 9 − − − IgG/ IgG/ 1,790 1,790 Fc Fc10 + − − IgG/ IgG/ 1,630 — Fc Fc 11 + − − — IgG/ 2,320 1,393 Fc 12 + − −— IgG/ — 5,510 Fc 13 + − − IgG/ IgG/ 1,500 1,315 Fc Fc 14 + − − — IgG/ —107 Fc; Fab 15 + + − IgG/ IgG/ 4,450 2,620 Fab; Fc Fc 16 + − + IgG — —1,099

The ADA immunoassay as reported in Example 5 was used to analyze 148different serum samples from rheumatoid arthritis patients taken atbaseline and after administration of tocilizumab. The results of theanalysis with the interference-suppressed ADA assay were compared withthose obtained by analysis with the conventional ADA immunoassay. Formore detailed analysis, a total of 92 serum samples (out of the 148)from 18 different patients with additional information on ADA isotypeand binding region as well as on clinical events were selected. Patientswere selected if they fulfilled at least one of the followingcriteria: 1) ADA positive immune response at any time point; 2) high TCZserum concentration; 3) clinical reaction such as infusion-related,hypersensitivity or anaphylaxis. Analysis of the binding region and theisotype of the ADAs were performed with a biosensor immunoassay aspreviously described (Stubenrauch, K., et al., Anal. Biochem. 390 (2009)189-196). Briefly, the surface plasmon resonance (SPR) assay set up madeuse of the four parallel flow cells on a single biosensor chip byimmobilization of full-length antibody and its constant (Fc) and antigenbinding (Fab) fragments for differential binding analysis of ADAs. Thepositive control standard conjugates mimicking polyclonal human ADAs ofdifferent isotypes were obtained by conjugating polyclonal rabbitantibodies against TCZ to human immunoglobulin (Ig) M, IgG, or IgE (seeWO 2008/061684). The Rheumatoid Factor (RF) assay was performed on theSiemens

BN II Nephelometer using RF reagents from Siemens Healthcare Diagnostics(Newark, Del., USA). Briefly, polystyrene particles coated with animmune-complex consisting of human immunoglobulin and anti-human IgGfrom sheep are aggregated when mixed with samples containing RF. Theseaggregates scatter a beam of light passed through the sample. Theintensity of the scattered light is proportional to the concentration ofthe respective protein in the sample. The result is evaluated bycomparison with a standard of known concentration.

Comparative evaluation of 258 different serum samples from TCZ-treatedRA patients with the conventional and interference-suppresseddrug-tolerant ADA assay as reported herein showed the same positiveresults in 12 Samples. The conventional assay measured 27 placebopatients positive whereas the interference-suppressed drug-tolerant ADAassay as reported herein 4. In conclusion, the set of measures asdescribed herein and the addition of oligomeric human IgG as an ADAassay additive conferred increased drug tolerance and suppressedinterference by RF compared to the conventional ADA assay.

A subset analysis of above-mentioned data is shown in Table 7 below.

TABLE 7 interference- conventional suppressed Patient Time point ELISAELISA dosing P1 week 4 + 0.812 + 0.110 placebo P2 baseline + 1.009 +0.893 4 mg/kg P2 week 4 − 0.097 + 0.088 4 mg/kg P2 week 9 + 0.276 +0.228 4 mg/kg P3 week 8 + 0.349 + 0.566 4 mg/kg P3 week 24 + 2.405 +1.760 4 mg/kg P3 week 28 + 1.307 + 1.001 4 mg/kg P4 week 4 + 1.409 +0.242 4 mg/kg P5 week 28 + 0.219 + 0.060 8 mg/kg P6 week 8 + 0.387 +0.688 4 mg/kg P6 week 12 + 3.689 + 3.721 4 mg/kg P6 week 24 + 3.506 +3.722 4 mg/kg P7 week 4 + 0.771 + 0.058 placebo study related CP: 0.215(conventional ELISA); 0.058 (interference-suppressed ELISA); +: positiveELISA result; −: negative ELISA result

In Table 8a assay signals for 27 placebo patients not treated with TCZare shown as bars and numbers. Due to the absence of TCZ-treatmentinduced ADA, high assay signals in this group are not expected and wouldindicate a potential interference.

TABLE 8a interference- conventional suppressed Patient Time point ELISAELISA dosing P8 baseline + 0.270 − 0.032 placebo P8 week 4 + 0.231 −0.033 placebo P9 week8 + 3.736 − 0.055 placebo P9 baseline + 3.297 +0.058 placebo P9 week 12 + 2.863 − 0.046 placebo P9 week 4 + 3.739 −0.052 placebo P1 week 4 + 0.812 + 0.110 placebo P10 week 4 + 0.755 −0.021 placebo P10 week 4 + 0.635 − 0.021 placebo P11 baseline + 0.272 −0.020 placebo P11 week 4 + 0.271 − 0.021 placebo P11 week 4 + 0.234 −0.020 placebo P12 baseline + 1.157 − 0.027 placebo P12 baseline + 1.362− 0.028 placebo P12 week 4 + 1.149 − 0.029 placebo P4 baseline + 0.522 −0.033 placebo P4 week 4 + 1.409 + 0.242 placebo P4 week 4 + 0.651 −0.047 placebo P13 week 36 + 0.349 − 0.023 placebo P14 week 4 + 0.245 −0.033 placebo P14 baseline + 0.276 − 0.035 placebo P14 week 4 + 0.275 −0.037 placebo P15 week 4 + 0.580 − 0.049 placebo P7 baseline + 0.990 −0.056 placebo P7 week 4 + 0.822 − 0.052 placebo P7 week 4 + 0.523 −0.042 placebo P7 week 4 + 0.771 + 0.058 placebo study related CP: 0.215(conventional ELISA); 0.058 (interference-suppressed ELISA); +: positiveELISA result; −: negative ELISA result

Signal pattern of both assay are very different: whereas all 27 placebossamples were determined to be positive using the conventional ELISA,only 4 out of the 27 sample were determined to be positive with theinterference-suppressed ELISA as reported herein.

In addition to placebo-treated patients sample of TCZ-treated patientshave been analyzed. In Table 8b results for the patients prior toTCZ-treatment are shown. In Table 8c results for TCZ-treated patientsare shown.

TABLE 8b interference- conventional suppressed Patient Time point ELISAELISA dosing P2 baseline + 1.009 + 0.893 4 P16 baseline + 0.745 − 0.0218 P17 baseline + 0.281 − 0.020 8 P18 baseline + 1.401 − 0.023 4 studyrelated CP: 0.215 (conventional ELISA); 0.058 (interference-suppressedELISA); +: positive ELISA result; −: negative ELISA result

TABLE 8c interference- conventional suppressed Patient Time point ELISAELISA dosing P19 week 24 + 0.281 − 0.030 4 mg/kg P19 week 24 + 0.226 −0.031 4 mg/kg P19 week 4 + 0.293 − 0.030 4 mg/kg P19 week 8 + 0.362 −0.028 4 mg/kg P2 week 4 − 0.097 + 0.088 4 mg/kg P2 week 8 + 0.276 +0.228 4 mg/kg P16 week 24 + 0.416 − 0.030 8 mg/kg P16 week 4 + 0.542 −0.029 8 mg/kg P16 week4 + 0.397 − 0.024 8 mg/kg P3 week 24 + 2.405 +1.760 4 mg/kg P3 week 28 + 1.307 + 1.001 4 mg/kg P3 week 4 − 0.181 +0.120 4 mg/kg P3 week 8 + 0.349 + 0.566 4 mg/kg P20 week 8 + 0.818 −0.034 4 mg/kg P21 week 4 + 0.289 − 0.031 8 mg/kg P17 week 12 + 0.369 −0.023 8 mg/kg P17 week 24 + 0.330 − 0.022 8 mg/kg P17 week 4 + 0.488 −0.022 8 mg/kg P17 week 4 + 0.465 − 0.025 8 mg/kg P17 week 8 + 0.409 −0.026 8 mg/kg P18 week 4 + 1.218 − 0.026 4 mg/kg P18 week 4 + 1.041 −0.028 4 mg/kg P5 week 28 + 0.219 + 0.060 8 mg/kg P6 week 12 + 3.689 +3.721 4 mg/kg P6 week 24 + 3.506 + 3.722 4 mg/kg P6 week 8 + 0.387 +0.688 4 mg/kg P22 week24 + 0.423 − 0.020 4 mg/kg study related CP: 0.215(conventional ELISA); 0.058 (interference-suppressed ELISA); +: positiveELISA result; −: negative ELISA result

The signal pattern with both assays is not similar: whereas 25 patientswere determined to be positive using the conventional ELISA only 10patients were determined to be positive using theinterference-suppressed ELISA as reported herein.

Example 7 Influence of Kind of Derivatization of Capture and TracerReagents

Mono- vs. Multi-Labeling

Measures to increase drug tolerance consisting in increasing theconcentration of biotinylated and digoxigenylated TCZ (e.g. to 1.5μg/mL); this could cause higher background by sticky digoxigenin; Tobeware of worse drug tolerance due to higher cut-points, a monobiotinylation and mono digoxigenylation give lower background signals bypresence of higher capture and tracer concentration.

A sandwich ELISA was used for both screening and confirmation ofanti-drug antibodies (ADAs) against tocilizumab (TCZ) (see Stubenrauch,K., et al., Clin. Ther. 32 (2010) 1597-1609). The principle of themethod is the capture of ADAs in complex with TCZ-Dig(mono) andTCZ-Bi(mono), the latter one leading to immobilization onto astreptavidin-coated plate. The TCZ-Bi/ADA/TCZ-Dig complex(pre-incubation overnight) bound to the SA-MTP was detected by ananti-Dig-HRP enzyme conjugated antibody. The principle of thedrug-tolerant anti-drug antibody assay is shown in FIG. 1. Inclusion ofoligomeric IgG as ADA assay additive leads to an interference-suppressedanti-drug antibody assay as shown in FIG. 2. The horseradish peroxidase(HRP) of the polyclonal antibody catalyzes a color reaction of thesubstrate ABTS. The color intensity is proportional to the concentrationof the analyte.

The screening assay was performed at room temperature. Reagents andserum samples were diluted with Universal buffer (cat no. 4742672), allwashing steps were performed with the washing buffer (PBS, 0.05%polysorbate 20 (Tween® 20) (cat. no. 11332465-001)) three times with 300μL per well. Incubations were performed under shaking on a microtiterplate shaker (MTP shaker) at 500 rpm. Test samples, QC and CS sampleswere incubated overnight (16 hours) with the capture antibody TCZ-Bi andthe detection antibody TCZ-Dig. Each well of the pre-incubationmicrotiter plate (MTP) was loaded with 225 μL of the capture/detectionsolution containing 1.667 μg/mL TCZ-Bi(mono) and 1.667 μg/mL TCZ-Dig(mono) with oligomeric human IgG ADA assay additive (AAA) and thereafter25 μL of the respective samples were added. The resulting concentrationsof TCZ-Bi(mono) and TCZ-Dig(mono) were 1.5 μg/mL each and the oligomerichuman IgG had a concentration of 50 μg/mL. The loaded MTP was covered toprevent evaporation and incubated overnight. Duplicates of 100 μL ofeach well of the pre-incubation plate were transferred to the wells of astreptavidin-coated microtiter plate (SA-MTP) which was covered andincubated for 1 h.

After washing, the polyclonal anti-Dig Fab-HRP conjugate with aconcentration of 25 mU/mL was added in a volume of 100 μL to each welland incubated for 1 h. After washing, the ABTS ready-to-use solution wasadded in 100 μL aliquots to each well and incubated for about 10 to 15min. while shaking. The signal of the color-generating reaction wasmeasured by an ELISA reader at 405 nm wavelength (reference wavelength:490 nm). Absorbance values of each serum sample were determined intriplicates. The highest standard should reach an optical density (OD)between 1.8 and 2.2 arbitrary units (AU). The obtained OD data was usedfor generating the standard calibration curve by non-linear 4-paramterfit “Wiemer Rodbard” for calculating the sample concentration. A samplewas confirmed as positive to ADAs if the recovery of the concentrationwas less than the specificity cut-point.

To determine a cut point 35 native sera of patients with RD (rheumaticdisease) were measured in both assays. As shown in FIGS. 5 and 6 thevariances between the signals of the sera in the interference-suppressedanti-drug antibody ELISA compared to the interference-suppressedanti-drug antibody ELISA with TCZ-Bi(mono) and TCZ-Dig(mono) are high.

For further assessment 77 different serum samples from TCZ-treated RApatients were analyzed with both variants of interference-suppressedADA-assay. Because all samples were taken before TCZ treatment(baseline), ADA against TCZ should be absent. Signals higher than cutpoint may indicated inference (false positives) not related with ADAagainst the treatment drug (TCZ),

The assay with multi-labeled TCZ (FIG. 7) showed more signal variationthan the assay with mono labeled TCZ (FIG. 8). As can be seen thisvariation is not a systemic setup of the system, which would simply be ashift on the Y-axis, but it is an increase in the bandwidth of obtainedsignals. Using these assay-specific cut points nearly all samples in theinterference-suppressed drug-tolerant ADA assay (mono) are below cutpoint. Mostly all samples measured in the multi assay are above the cutpoint.

What is claimed is:
 1. A method for the detection of anti-drugantibodies against a drug antibody in a sample of an rheumatoidarthritis patient comprising a capture drug antibody and a tracer drugantibody, wherein a) the capture drug antibody and the tracer drugantibody have a concentration of 0.5 μg/ml or more, b) the sample isincubated simultaneously with the capture drug antibody and the tracerdrug antibody for 0.5 to 24 hours, c) the capture drug antibody and thetracer drug antibody are each derivatized only once via a single lysineresidue, d) the sample comprises 1% serum/plasma or more, e) oligomerichuman IgG is added to the sample prior to the incubation with thecapture drug antibody and the tracer drug antibody, and f) monomericbiotin and digoxygenin on capture and tracer.
 2. The method according toclaim 1, wherein the sample comprises anti-drug antibodies andrheumatoid factors.
 3. The method according to claim 1, wherein theantibody is an anti-inflammatory antibody.
 4. The method according toclaim 3, wherein the anti-inflammatory antibody is an antibody for thetreatment of rheumatoid arthritis, or juvenile arthritis, orosteoarthritis, or Castleman's disease, or mesangial proliferativeglomerulonephritis.
 5. The method according to claim 1, wherein theantibody is an anti-cancer antibody.
 6. The method according to claim 5,wherein the anti-cancer antibody is an antibody for the treatment ofmyeloma or plasmacytoma.
 7. The method according to claim 1, wherein theantibody is an anti-IL6R antibody.
 8. The method according to claim 7,wherein the anti-IL6R antibody is tocilizumab.
 9. A method of treatingan individual having plasmacytoma comprising administering to theindividual an effective amount of an anti-IL6R antibody (drug) anddetermining the presence of anti-anti-IL6R antibody antibodies(anti-drug antibodies) with an assay according to claim
 1. 10. A methodof treating an individual having myeloma comprising administering to theindividual an effective amount of an anti-IL6R antibody (drug) anddetermining the presence of anti-anti-IL6R antibody antibodies(anti-drug antibodies) with an assay according to claim
 1. 11. A methodof inhibiting IL6R activity in an individual comprising administering tothe individual an effective amount of an anti-IL6R antibody to inhibitIL6R activity and determining the presence of anti-anti-IL6R antibodyantibodies (anti-drug antibodies) with an assay according to claim 1.